Nucleic acid recombination in vitro lies at the core of molecular biology and biotechnology. The efficiency whereby recombinant nucleic acid technology is achieved can dictate the outcome of certain biotechnology implementations. For example, the cloning of recombinant DNA molecules is dramatically enhanced by several orders of magnitude when the overlapping fragments are annealed together via sticky ends and ligated with a ligase enzyme in vitro as compared to blunt-ended fragments ligated under similar condition. Likewise, fragments having significant overlap (for example, G/C- or A/T-tailed fragments) can result in successful transformation and cloning of recombinant fragments without ligation in vitro prior to transformation, wherein the resultant transformed molecules are repaired and ligated together inside cells following transformation. Yet the use of ligase reactions in vitro to form joined products prior to transformation dramatically increases the efficiency of cloning molecules containing overlap regions.
More complex recombinant ligations, such as the assembly and ligation of a plurality of recombinant fragments into a vector, necessitates the use of ligase reactions to ensure acceptable cloning efficiencies of the resultant recombinant products. In one implementation, Gibson et al. describe in U.S. Pat. No. 7,776,532 (“the '532 patent”), entitled METHOD FOR IN VITRO RECOMBINATION, a method for directed assembly and ligation of a plurality of recombinant fragments. The method relies on the use of isolated protein reagents for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity. The first step includes chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule that contain a sufficient length of the region of sequence identity to hybridize specifically to each other. The second step includes specifically annealing the single-stranded overhangs. The third step includes repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (that is, ligating the nicked DNA molecules). The region of sequence identity generally typically includes at least 20 non-palindromic nucleotides (nt), such as at least about 40 non-palindromic nt. A crowding agent is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, for example, to join synthetically produced sub-fragments of a gene or genome of interest.
Crowding Agents
The '532 patent defines a crowding agent as a compound that allows for, enhances or facilitates molecular crowding. The '532 patent provides examples of crowding agents as macromolecular polymer species such as polyethylene glycol (PEG 200 and up, including 20,000 and up); Ficoll, such as Ficoll 70; dextran, such as dextran 70; or the like. The '532 patent teaches that a crowding agent provides molecular crowding to bind to and tie up water in a solution, thereby removing water from components suspended in solution to allow components of the solution to come into closer contact with one another.
The '532 patent teaching about crowding agents is also consistent with and supported by that found in the literature. Bhat R & Timasheff S N in Protein Sci. 1992 1:1133-43 describe the crowding effect of polyethylene glycol on proteins by affecting the hydration state of the proteins. Hatters D M et al. in J. Biol. Chem. 2002 277:7824-30 examine the effect of excluded volume on aggregation using concentrated solution of dextran. Minton A P in J. Biol. Chem. 2001 276:10577-80 reviews the effect of crowding on enzyme activity. Sanders G M et al. in Proc. Natl. Acad. Sci. U.S.A. 1994 91:7703-7 describe the use of the crowding agents polyethylene glycol, polyvinyl alcohol, dextran, and Ficoll to enhance in vitro transcription.
Chaperone Agents
The literature teaches a class of small molecules having chaperone activity that differ from the function of crowding agents, such as those described in the '532 patent. The literature suggests that chaperone agents have the opposite affects of crowding agents, as described below.
Anderson, J A, in J. Am. Soc. Hort. Sci. 2007 132:67-72 describes polyols, as well as sugars, amino acids and methylamines as chemical chaperones that stabilize proteins in response to stress such as high temperature. Anderson provides examples of chemical chaperones having such activity to include mannitol, glycerol, trehalose, maltose, sucrose, glycine, betaine and trimethylamine.
Bounedjah O. et al. in J. Biol. Chem. 2012 287:2446-58 states that osmolytes (that include chaperones) serve a protective function and reduce the effects of macromolecular crowding. These authors identify betaine, taurine, and myo-inositol as having such protective functions to reducing macromolecular crowding.
Ghahghaei A et al. in Int. J. Peptide Res. Therap. 2011 17:101-11 states that glycerol is a chemical chaperone belonging to the polyol family that increases protein stability and inhibits protein aggregation. These authors describe β-casein as a molecular chaperone and differentiate these compounds from crowding agents such as dextran.
Levy-Sakin M et al. in PLoS One 2014 9:e88541 describes glycerol and other polyols as chaperones and protein stabilizers. These authors state that small carbohydrates have the same effect and studied the ability of ethylene glycol, glycerol, D-theritol, meso-erythritol, D-adonitol, L-arabitol, xylitol, D-sorbitol and D-mannitol to stabilize the activity of trypsin under stress.
Mishra R et al. in J. Biol. Chem. 2005 280:15553-60 describe the ability of glycerol to suppress protein aggregation. These authors define polyethylene glycol as a “viscogen” that suppresses protein folding (that leads to aggregation).
Moody M et al. in 1st Tabriz Intl. Life Sci. Conf & 12th Iran Biophys. Chem. Conf. state that glycerol and arginine are both chemical chaperones known to stabilize protein conformation and prevent aggregation in a crowded environment created by the presence of dextran.
Olsen S N, et al. in Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2007 148:339-45. These authors studied the effect of osmolytes on enzyme activity in combination with macromolecular crowding.
Perlmutter D H in Pediatr Res. 2002 52:832-6 describes polyols (glycerol), trimethylamines and amino acid derivatives as chemical chaperones.
Sukenik S et al. in PLoS One 2011 6:e15608 shows that osmolytes stabilize the monomeric state of amyloid, but also stabilizes the fibril once it is formed.
Uversky V N in Protein J. 2009 28:305-25 shows that the osmolytes proline, sarcosine and sorbitol (a polyol) effectively induced protein folding.
Zancan P & Sola-Penna M. in Arch. Biochem. Biophys. 2005 444:52-60 demonstrates that trehalose and glycerol can function as chemical chaperones.
The '532 patent does not recite that crowding agents include chaperones and osmolytes or examples of the same, such as glycerol and the other small molecules describe in the literature. For this reason—and owing to the possible confusing overlap between these apparently distinct classes of compounds having disparate effects on mixtures of nucleic acids and nucleic acid-modifying enzymes, there remains to be established whether small molecules acting as chaperones have any effect on nucleic acids and nucleic acid modifying enzymes directed to in vitro recombination of overlapping double-stranded fragments in assembling reactions.